Secondary metabolites – natural products produced by plants and microorganisms are a prolific source for chemicals, drugs, fuels, and materials. Recent advances in molecular biology and genomics have revolutionized our ability to discover the biosynthetic pathways that synthesize natural products. In this talk, I will present two of our recent works on discovery, characterization, and engineering of new natural products. The first example concerns phloroglucinol, a precursor that is used to produce a variety of high value bioactive compounds and energetic compounds and is currently produced at 140 tons/yr using chemical methods. We discovered a novel type III polyketide synthase, PhlD, from Pseudomonas fluorescens that enables the direct biosynthesis of phloroglucinol from D-glucose. Heterologous expression of PhlD in E. coli led to the production of phloroglucinol in vivo, with an estimated amount of 0.7 g/L under the shaking flask condition and 10 g/L by using a continuous fermentation. To further improve the phloroglucinol yield, directed evolution was applied to enhance the activity of PhlD, which is facilitated by a high through-put screening to assay the improvement in phloroglucinol production. In addition, metabolic pathway engineering was carried out to redirect the carbon flux inside E. coli to pathways responsible for the synthesis of phloroglucinol. The second example concerns FR900098, a novel chemotherapeutic agent for the treatment of malaria. We cloned the FR-900098 biosynthetic gene cluster from Streptomyces rubellomurinus and heterologously expressed it in Streptomyces lividans. In addition, we constructed an artificial gene cluster that contains the FR900098 biosynthetic genes all under the control of individual T7 promoters in E. coli. The various biosynthetic steps were characterized by gene deletions, in vivo feeding experiments, and in vitro enzymatic assays. Several novel enzymatic reactions were uncovered. The resulting recombinant E. coli strain produced FR900098 at 6 mg/L. Metabolic engineering strategies aimed at optimizing gene expression and increasing precursors and cofactors are also explored.

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